One-Step Construction of Allogenic Car-Nk Cells with Increased Anti-Tumor Cytotoxicity and Resistance to Host Cell Rejection

This application claims priority to and the benefit of U.S. Provisional Application No. 63/568,664, filed on Mar. 22, 2024, which is incorporated by reference herein in its entirety.

The present disclosure relates to RNAi DNA oligonucleotides for the suppression of an immune response and use in methods of one step construction of allogenic CAR cells capable of avoiding a host rejection.

The sequence listing associate with this application is provided in XML format in lieu of a paper copy and is hereby incorporated by reference into the specification. The name of the XML file containing the sequence listing is “MIT25757J_SeqListing.xml”. The XML file is 32,800 bytes, and created on Mar. 18, 2025, and is being submitted electronically, concurrent with the filing of this specification.

Chimeric antigen receptor (CAR) cell therapy is one of the most promising approaches in anti-cancer treatments. The recent approvals of CD19 and BCMA targeting CAR-T cell therapies are significant breakthroughs in the field of cancer immunotherapy and have stimulated the development of genetically modified cell therapies for both hematological malignancies and solid tumors (Xie et al., 2020). To date, all six approved CAR-T cell therapies use patients' own (autologous) T cells for manufacturing (Dephil et al., 2020). However, autologous CAR cell therapies have well-known drawbacks, including 1) high cost and long vein-to-vein time due to the requirement of individualized manufacturing of CAR cells (Köhl et al., 2018; Laskowski et al., 2022); 2) the autologous CAR cells might not be effective in some patients owing to the dysfunction of source immune cells, which have been continuously stimulated by cancer cells and suppressed in the tumor microenvironment (Thommen & Schumacher, 2018); and 3) many patients have gone through multiple previous lines of treatment and are lymphopenic, leading to insufficient immune cells to yield a viable product (Laskowski et al., 2022).

The capability of using immune cells from any healthy donors (allogeneic) could potentially solve these issues. However, allogeneic cell therapies need to overcome two major issues: avoid graft-versus-host disease (GVHD) and rejection of allogeneic CAR cells. Unlike T cells, which can recognize non-self peptide/MHC and induce GVHD, natural killer (NK) cells are modulated by a set of activating and inhibitory receptors and don't mediate allo-reaction (Xie et al., 2020). Hence, NK cells have advantages over T cells in allogeneic cell therapies. However, allogeneic NK cells can be recognized and rapidly rejected by the recipient's immune system.

Human leukocyte antigen (HLA) class I molecules play central roles in modulating the T cell and NK cell responses, and it's an important consideration for allogeneic cell therapy (Montgomery et al., 2018). Classical HLA class I, including HLA-A, HLA-B, and HLA-C, (referred to herein as “HLA-ABC”) presents non-self and neoantigen-derived intracellular peptides on the cell surface to CD8+ T cells and subsequently activates their functions (Neefjes et al., 2011). However, non-classical HLA class I, HLA-E, mainly presents peptides derived from the leader sequences of HLA-ABC and HLA-G on the cell surface and interacts with CD94/NKG2 family receptors to modulate NK cell functions (Braud et al., 1998). HLA class I molecules consist of three components: the polymorphic heavy chain, the light chain β2-microglobulin (β2m), and the presented peptide (Neefjes et al., 2011). Of these three components, the β2m genes and those related to the peptide presentation-associated pathway, like transporter associated with antigen processing (TAP), are widely targeted for knocking out and knocking down HLA class I expression, and no doubt both HLA-ABC and HLA-E would be reduced. The HLA-ABC-reduced cells can escape from allogeneic T-cell killing, whereas they are susceptible to NK cell killing because NK cells recognize and kill HLA-ABC-reduced target cells (Anfossi et al., 2006). Thus, it's critical to specifically reduce surface HLA-ABC expression but maintain surface HLA-E expression.

Inhibitory immunoreceptors, also known as immune checkpoints, can suppress immune cells upon interacting with ligands on immune cells (He & Xu, 2020). The use of immune checkpoint inhibitors (ICIs) has significantly improved cancer treatment, enabling the possibility of long-term survival of patients with metastatic tumors (Johnson et al., 2022). Recently, immune checkpoint proteins were overexpressed on target cells to allow them to sustain longer in allogeneic hosts (Hu et al., 2023) showed that CD47 overexpressed primary human islets and induced pluripotent stem cells evaded NK and macrophage killing, and MHC I/II knockout and CD47 overexpressed cells survived in allogeneic recipients for a long term (Hu et al., 2023a,b). Yoshihara et al. demonstrated that overexpression of PD-L1 protected human islet-like organoids, resulting in the restoration of glucose homeostasis in immune-competent diabetic mice for 50 days (Yoshihara et al., 2018). In addition, Gornalusse et al. found that HLA-E overexpressing and HLA-ABC negative pluripotent stem cells escape from CD8+ T cell killing and lysis by NK cells in vitro and in vivo (Gornalusse et al., 2017).

Thus, there is a need for development of allogenic cell therapies that avoid rejection by host immune cells.

One embodiment described herein is an RNA interference (RNAi) DNA oligonucleotide for inhibiting expression of HLA A, B and C. In one aspect, the RNAi DNA oligonucleotide comprises siRNA or shRNA. In another aspect, the RNAi DNA oligonucleotide does not inhibit expression of HLA-E. In another aspect, the RNAi DNA oligonucleotide is shRNA.

In another aspect, the RNAi DNA oligonucleotide is 20-25 base pairs long. In another aspect, the RNAi DNA oligonucleotide comprises at least two mismatches. In one aspect, the RNAi DNA oligonucleotide comprises three mismatches. In another aspect, the mismatches are with an allele comprising HLA-A, HLA-B, HLA-C, or HLA-E. In another aspect, the mismatch is with an HLA-E allele.

In another aspect, the RNAi DNA oligonucleotide comprises a nucleotide sequence of SEQ ID NO. 1-30 and its complement. In one aspect, the RNAi DNA oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 13, and its complement.

Another aspect is a vector comprising the RNAi DNA oligonucleotide described herein. In another aspect, the vector is a lentiviral vector. In another aspect of the vector, the RNAi DNA oligonucleotide is operably linked to a promoter. In another aspect of the vector described herein, the promoter comprises an RNA polymerase III or an EF-1α promoter.

In another aspect, the vector further comprises a nucleotide sequence encoding HLA-E or variants thereof. In another aspect, the HLA-E sequence comprises a mutation in the heavy chain. In another aspect, the HLA-E mutation comprises Y84A or Y84C. In another aspect, the vector further comprises a nucleotide sequence encoding PD-L1.

Another aspect is a host cell transduced by the vector described herein. In another aspect, the host cell is a T cell, a Natural Killer (NK) cell, a macrophage, or an induced pluripotent stem cell.

Another embodiment described herein is a method of making an allogenic chimeric antigen receptor (CAR) immune cell resistant to host rejection comprising transducing an immune cell with the vector described herein. In one aspect of the method, the vector further comprises a nucleotide sequence encoding HLA-E or variants thereof. In one aspect of the method, the vector further comprises a nucleotide sequence encoding PD-L1. In one aspect of the method, the vector comprises the nucleotide sequence of SEQ ID NO. 1 or SEQ ID NO: 13, and its complement, a nucleotide sequence encoding HLA-E or variants thereof, and a nucleotide sequence encoding PD-L1, operably linked to one or more heterologous promoter. In another aspect of the method, the cell is a T cell, an NK cell, a macrophage, or an induced pluripotent stem cell.

Another embodiment described herein is a method of making an allogenic cell resistant to host rejection comprising transducing the cell with a vector comprising a nucleotide sequence encoding at least one of HLA-E or variant thereof, or PD-L1.

Another embodiment described herein is a method for increasing cytotoxicity of a CAR cell.

Chimeric antigen receptor (CAR)-armed T cells (CAR-T) and natural killer cells (CAR-NK) have demonstrated great efficacy in treating liquid tumors and are being developed for treating solid tumors. Typically, a patient's own T cells or NK cells are harvested and transduced with lentivectors encoding CAR, expanded in vitro, and then transferred back into the same patients for cancer therapy. There is a great need to transduce allogeneic T cells and NK cells from healthy donors for cancer therapy because T cells and NK cells from healthy donors are easier to source and more active and can be prepared as “off-the-shelf” products. However, allogeneic T cells and NK cells are rejected by host immune cells, including T cells and NK cells. To suppress the rejection of allogenic CAR-T and CAR-NK cells, one common approach is to inhibit MHC class I expression on allogenic T and NK cells

Described herein are novel RNAi DNA oligonucleotides for suppression of HLA-ABC, a method of making an allogenic chimeric antigen receptor (CAR) immune cell resistant to host cell rejection, and a method for increasing cytotoxicity of a chimeric antigen receptor (CAR) cell.

While various embodiments of the disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed.

It is to be understood that the methods described in this disclosure are not limited to particular methods and experimental conditions disclosed herein; as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. Furthermore, the experiments described herein, unless otherwise indicated, use conventional molecular and cellular biological and immunological techniques within the skill of the art. Such techniques are well known to the skilled worker, and are explained fully in the literature. See, e.g., Ausubel, et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., Ny, Ny. (1987-2008), including all supplements, Molecular Cloning: A Laboratory Manual (Fourth Edition) by MR Green and J. Sambrook.

Generally, nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques provided herein are generally performed according to convention methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated.

Unless otherwise defined herein, scientific and technical terms used herein have the meanings that are commonly understood by those of ordinary skill in the art. In the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The use of “or” means “and/or” unless stated otherwise. The use of the term “including”, as well as other forms, such as “includes” and “included”, is not limiting.

Unless otherwise defined, all terms of art, notations, and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which the application pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. The following references provide one of skill with a general definition of many of the terms used in the instant disclosure: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994): The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, the Harper Collins Dictionary of Biology (1991).

As used herein, the following terms have the meanings as ascribed to them below, unless specified otherwise.

Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms “a”, “an”, and “the” are understood to be singular or plural.

Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.

As used herein, the term “antisense oligonucleotide” means a plurality of linked nucleosides, at least a portion of which, is complementary to a target nucleic acid to which it is capable of hybridizing, resulting in at least one antisense activity. In one aspect described herein, oligonucleotides comprise one or more of deoxyribonucleosides (DNA) and/or ribonucleosides (RNA). As used herein a “nucleotide” means a nucleoside further comprising a phosphate linking group. The nucleotides described herein may be found in both DNA and RNA, and may be referred to by their full name, or single letter abbreviation, all interchangeably.

The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” may mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” may mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. In another example, the amount “about 10” includes 10 and any amounts from 9 to 11. In yet another example, the term “about” in relation to a reference numerical value may also include a range of values plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from that value. Alternatively, particularly with respect to biological systems or processes, the term “about” may mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.

As used herein, the term “chimeric antigen receptor cell” means any immune cell which is genetically modified to express a chimeric receptor with specificity for a particular antigen. Exemplary immune cells include, but are not limited to, T-cells, Natural Killer (NK) cells, pluripotent stem cells or macrophages. Cells may have specificity for any known antigen, including but not limited to, CD19, BCMA, and Mesothelin specific CAR cells. CARs may include (1) an extracellular antigen-binding motif (e.g., single-chain variable fragment (scFv) antibody), (2) linking/transmembrane motifs, and (3) an intracellular domain, including a costimulatory domain and an activity domain (e.g., CD137 (4-IBB) and CD247 (CD3ζ)-derived costimulatory domain and an activity domain, respectively). In aspects, the CAR expresses an anti-mesothelin antibody or fragment thereof or an anti-CD19 antibody or fragment thereof (e.g., Intl. Pat. App. No. PCT/US2023/066794, filed May 9, 2023, incorporated herein by reference in its entirety; US. Pat. App. No. U.S. Ser. No. 17/910,776, filed Jan. 8, 2021, incorporated herein by reference in its entirety).

As used herein, the term “complementary” in reference to oligonucleotides means the capacity of the oligonucleotide to hybridize to another oligonucleotide compound or region via established Watson-Crick nucleotide base pairing rules, resulting in hybridization. Some mismatches are tolerated, thus in one aspect, antisense oligonucleotides may be 70% complementary. In other aspects, antisense oligonucleotides may be 80% complementary. In some aspects, antisense oligonucleotides may be 90% complementary. In some aspects, antisense oligonucleotides may be 95% complementary. In yet other aspects, antisense oligonucleotides may be 99% complementary. In yet other aspects, antisense oligonucleotides may be 100% complementary.

The term “comprise”, “comprises”, and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. By “consisting of” is meant to include, and be limited to, whatever follows the phrase “consisting of.” Thus, the phrase “consisting of” indicates that the listed elements are required or mandatory, and that no other elements may be present. By “consisting essentially of” is meant including any elements listed after the phrase, and be limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements.

An “expression vector” or “vector” is any genetic element, e.g., a plasmid, a mini-circle, a nanoplasmid, chromosome, virus, transposon, behaving either as an autonomous unit of polynucleotide replication within a cell. (i.e. capable of replication under its own control) or being rendered capable of replication by insertion into a host cell chromosome, having attached to it another polynucleotide segment, so as to bring about the replication and/or expression of the attached segment. Suitable vectors include, but are not limited to, plasmids, transposons, bacteriophages, cosmids or virus based vectors. Vectors may contain polynucleotide sequences which are necessary to effect ligation or insertion of the vector into a desired host cell and to effect the expression of the attached segment. Such sequences differ depending on the host organism; they include promoter sequences to effect transcription, enhancer sequences to increase transcription, ribosomal binding site sequences and transcription and translation termination sequences. Alternatively, expression vectors may be capable of directly expressing nucleic acid sequence products encoded therein without ligation or integration of the vector into host cell DNA sequences. In some embodiments, the vector is an “episomal expression vector” or “episome,” which is able to replicate in a host cell, and persists as an extrachromosomal segment of DNA within the host cell in the presence of appropriate selective pressure (see, e.g., Conese et al., Gene Therapy, 11:1735-1742 (2004)). Representative commercially available episomal expression vectors include, but are not limited to, episomal plasmids that utilize Epstein Barr Nuclear Antigen 1 (EBNA1) and the Epstein Barr Virus (EBV) origin of replication (oriP). The vectors pREP4, pCEP4, pREP7, and pcDNA3.1 from Invitrogen (Carlsbad, Calif.) and pBK-CMV from Stratagene (La Jolla, Calif.) represent non-limiting examples of an episomal vector that uses T-antigen and the SV40 origin of replication in lieu of EBNA1 and oriP.

The term “promoter” refers to a region of a polynucleotide that initiates transcription of a coding sequence. Promoters are located near the transcription start sites of genes, on the same strand and upstream on the DNA (towards the 5′ region of the sense strand). Some promoters are constitutive as they are active in all circumstances in the cell, while others are regulated becoming active in response to specific stimuli, e.g., an inducible promoter. The term “promoter activity” and its grammatical equivalents as used herein refer to the extent of expression of nucleotide sequence that is operably linked to the promoter whose activity is being measured. Promoter activity may be measured directly by determining the amount of RNA transcript produced, for example by Northern blot analysis or indirectly by determining the amount of product coded for by the linked nucleic acid sequence, such as a reporter nucleic acid sequence linked to the promoter.

The term “operably linked” as used herein refers to refers to the physical and/or functional linkage of a DNA segment to another DNA segment in such a way as to allow the segments to function in their intended manners. A DNA sequence encoding a gene product is operably linked to a regulatory sequence when it is linked to the regulatory sequence, such as, for example, promoters, enhancers and/or silencers, in a manner, which allows modulation of transcription of the DNA sequence, directly or indirectly. For example, a DNA sequence is operably linked to a promoter when it is ligated to the promoter downstream with respect to the transcription initiation site of the promoter, in the correct reading frame with respect to the transcription initiation site and allows transcription elongation to proceed through the DNA sequence. An enhancer or silencer is operably linked to a DNA sequence coding for a gene product when it is ligated to the DNA sequence in such a manner as to increase or decrease, respectively, the transcription of the DNA sequence. Enhancers and silencers may be located upstream, downstream or embedded within the coding regions of the DNA sequence. A DNA for a signal sequence is operably linked to DNA coding for a polypeptide if the signal sequence is expressed as a pre-protein that participates in the secretion of the polypeptide. Linkage of DNA sequences to regulatory sequences is typically accomplished by ligation at suitable restriction sites or via adapters or linkers inserted in the sequence using restriction endonucleases known to one of skill in the art.

“Polynucleotide” or “oligonucleotide” as used herein refers to a polymeric form of nucleotides or nucleic acids of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Thus, this term includes double and single stranded DNA, triplex DNA, as well as double and single stranded RNA. It also includes modified, for example, by methylation and/or by capping, and unmodified forms of the polynucleotide. The term is also meant to include molecules that include non-naturally occurring or synthetic nucleotides as well as nucleotide analogs.

As used herein “RNAi DNA oligonucleotides” are double stranded DNA molecules for expression of an RNAi transcription product capable of sequence-specific suppression of gene expression at either the transcriptional or translational level. Exemplary RNAi DNA oligonucleotides described herein include, but are not limited to, small interfering RNA (siRNA) or short hairpin RNA (shRNA), both for expression of siRNA or shRNA transcription products. shRNA sequences may form stem-loops structure of 15 to 30 base pairs (bp) region of double-stranded RNA bridged by a single-stranded RNA “loop”. shRNAs may be subsequently cleaved at the loop by the nuclease Dicer in the cytoplasm, and enter the RISC to direct cleavage and subsequent degradation of complementary mRNA. In one aspect, an RNAi DNA oligonucleotide described herein comprises an siRNA or an shRNA. In another aspect, the RNAi DNA oligonucleotide may be about 15 to 22 bp, about 18 to 28 bp, about 19 to 27 bp, about 20 to 30 bps. In another aspect described herein is an RNAi DNA oligonucleotide means for inhibiting expression of HLA-A, HLA-B and HLA-C.

Unless otherwise stated, nucleic acid sequences in the text of this specification are given, when read from left to right, in the 5′ to 3′ direction.

As used herein, the phrase “variant” when used with reference to a nucleic acid or polypeptide refers to a nucleic acid or polypeptide that differs from the referenced nucleic acid or polypeptide (for example, differing by at least one amino acid substitution from a wild-type sequence) but possesses the primary function of the referenced polypeptide. For example, a functional variant of a polypeptide that serves as a transmembrane domain is a fragment of that polypeptide that also serves as a transmembrane domain. When used with reference to a nucleic acid, the phrase “variant” refers to a nucleic acid that differs from the referenced nucleic acid but encodes a polypeptide having the same primary function as the polypeptide encoded by the referenced nucleic acid.

The terms “transfection,” “transformation,” “nucleofection,” or “transduction” as used herein refer to the introduction of one or more exogenous polynucleotides into a host cell or organism by using physical, chemical, and/or electrical methods. The nucleic acid sequences and vectors disclosed herein may be introduced into a cell or organism by any such methods, including, for example, by electroporation, calcium phosphate co-precipitation, strontium phosphate DNA co-precipitation, liposome mediated-transfection, DEAE dextran mediated-transfection, polycationic mediated-transfection, tungsten particle-facilitated microparticle bombardment, viral, and/or non-viral mediated transfection. In some cases, the method of introducing nucleic acids into the cell or organism involve the use of viral, retroviral, lentiviral, or transposon, or transposable element-mediated (e.g., Sleeping Beauty) vectors. In another aspect described herein is a vector means for inhibiting expression of HLA-A, HLA-B and HLA-C.

“Polypeptide”, “peptide”, and their grammatical equivalents as used herein refer to a polymer of amino acid residues. The polypeptide may optionally include glycosylation or other modifications typical for a given protein in a given cellular environment. Polypeptides and proteins disclosed herein (including functional portions and functional variants thereof) may comprise synthetic amino acids in place of one or more naturally-occurring amino acids. Such synthetic amino acids are known in the art, and include, for example, aminocyclohexane carboxylic acid, norleucine, α-amino n-demayoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine p-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N′-benzyl-N′-methyl-lysine, N′,N′-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentane carboxylic acid, α-aminocyclohexane carboxylic acid, α-aminocycloheptane carboxylic acid, α-(2-amino-2-norbomane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine. The present disclosure further contemplates that expression of polypeptides or proteins described herein in an engineered cell may be associated with post-translational modifications of one or more amino acids of the polypeptide or protein. Non-limiting examples of post-translational modifications include phosphorylation, acylation including acetylation and formylation, glycosylation (including N-linked and O-linked), amidation, hydroxylation, alkylation including methylation and ethylation, ubiquitylation, addition of pyrrolidone carboxylic acid, formation of disulfide bridges, sulfation, myristoylation, palmitoylation, isoprenylation, farnesylation, geranylation, glypiation, lipoylation and iodination.

The terms “identical” and its grammatical equivalents as used herein or “sequence identity” in the context of two nucleic acid sequences or amino acid sequences of polypeptides refer to the residues in the two sequences which are the same when aligned for maximum correspondence over a specified comparison window. A “comparison window”, as used herein, refers to a segment of at least about 20 contiguous positions, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are aligned optimally. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman, Adv. Appl. Math., 2:482 (1981); by the alignment algorithm of Needleman and Wunsch, J. Mol. Biol., 48:443 (1970); by the search for similarity method of Pearson and Lipman, Proc. Nat. Acad. Sci U.S.A., 85:2444 (1988); by computerized implementations of these algorithms (including, but not limited to CLUSTAL in the PC/Gene program by Intelligentics, Mountain View Calif., GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis., U.S.A.); the CLUSTAL program is well described by Higgins and Sharp, Gene, 73:237-244(1988) and Higgins and Sharp, CABIOS, 5:151-153 (1989); Corpet et al., Nucleic Acids Res., 16:10881-10890 (1988); Huang et al., Computer Applications in the Biosciences, 8:155-165 (1992); and Pearson et al., Methods in Molecular Biology, 24:307-331 (1994). Alignment may also be performed by inspection and manual alignment. In one class of embodiments, the polypeptides herein are at least 80%, 85%, 90%, 98% 99% or 100% identical to a reference polypeptide, or a fragment thereof, e.g., as measured by BLASTP (or CLUSTAL, or any other available alignment software) using default parameters. Similarly, nucleic acids may also be described with reference to a starting nucleic acid, e.g., they may be 50%, 60%, 70%, 75%, 80%, 85%, 90%, 98%, 99% or 100% identical to a reference nucleic acid or a fragment thereof, e.g., as measured by BLASTN (or CLUSTAL, or any other available alignment software) using default parameters. When one molecule is said to have certain percentage of sequence identity with a larger molecule, it means that when the two molecules are optimally aligned, the percentage of residues in the smaller molecule finds a match residue in the larger molecule in accordance with the order by which the two molecules are optimally aligned.

The term “substantially identical” and its grammatical equivalents as applied to nucleic acid or amino acid sequences mean that a nucleic acid or amino acid sequence comprises a sequence that has at least 95% sequence identity with a reference sequence using the programs described above, e.g., BLAST, using standard parameters. For example, the BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=−4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1992)). Percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. In some embodiments, the substantial identity exists over a region of the sequences that is at least about 50 residues in length, over a region of at least about 100 residues, and in some embodiments, the sequences are substantially identical over at least about 150 residues. In some embodiments, the sequences are substantially identical over the entire length of the coding regions.

“Homology” is generally inferred from sequence identity between two or more nucleic acids or proteins (or sequences thereof). The precise percentage of identity between sequences that is useful in establishing homology varies with the nucleic acid and protein at issue, but as little as 25% sequence identity is routinely used to establish homology. Higher levels of sequence identity, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% or more may also be used to establish homology. Methods for determining sequence identity percentages (e.g., BLASTP and BLASTN using default parameters) are described herein and are generally available. Nucleic acids and/or nucleic acid sequences are “homologous” when they are derived, naturally or artificially, from a common ancestral nucleic acid or nucleic acid sequence. Proteins and/or protein sequences are “homologous” when their encoding DNAs are derived, naturally or artificially, from a common ancestral nucleic acid or nucleic acid sequence. The homologous molecules may be termed “homologs.” For example, any naturally occurring proteins may be modified by any available mutagenesis method. When expressed, this mutagenized nucleic acid encodes a polypeptide that is homologous to the protein encoded by the original nucleic acid.

Also contemplated and included herein are nucleic acid molecules that hybridize to the disclosed sequences. Hybridization conditions may be mild, moderate, or stringent, as is warranted. Appropriate stringency conditions which promote DNA hybridization, for example, 6× sodium chloride/sodium citrate (SSC) at about 450 C, followed by a wash of 2×SSC at 50° C., are known or may be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. “Stringent hybridization conditions” are those in which the salt concentration is less than about 1.5 M Na+ ion, typically about 0.01 to 1.0 M Na+ ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is at least about 30° C. for short sequences (such as, for example, 10 to 50 nucleotides) and at least about 60° C. for longer sequences (such as, for example, greater than 50 nucleotides). Optionally, wash buffers may comprise about 0.1% to about 1% SDS. Duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours. The duration of the wash time will be at least a length of time sufficient to reach equilibrium.

In one aspect described herein, a “host cell” includes cells transfected, infected, or transduced in vivo, ex vivo, or in vitro with a recombinant vector or a polynucleotide of the disclosure. Host cells may include packaging cells, producer cells, and cells infected with viral vectors. In some embodiments, host cells infected with viral vector of the disclosure are administered to a subject in need of therapy. In certain embodiments, the term “target cell” is used interchangeably with host cell and refers to transfected, infected, or transduced cells of a desired cell type. In some embodiments, the target cell is a T cell, an NK cell or an induced pluripotent stem cell (iPSC) cell. Host cells may be either autologous or allogenic. In some aspects, allogenic host cells may be obtained from a pool of healthy donors cells, including but not limited to, peripheral blood mononuclear cells (PMBCs), umbilical cord blood stem cells (UCBs), or induced pluripotent stem cell (iPSC) cells. In one aspect described herein, the host cell may be transduced with any of the vectors described herein. In another aspect is a host cell means for increased CAR cytotoxicity.

For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

Suppression of the immune response to allogenic CAR-T (and CAR-NK) therapy may be facilitated by suppression of HLA-ABC complex proteins. However, the immune response is multi-tiered, and includes not only T-cells, but NK cells and macrophages. Thus, while suppression of HLA-ABC may avoid the immune response from T-cells, attack from other cell types may be contemplated. NK cells in particular recognize and kill HLA-ABC-reduced cells. Thus, maintaining expression of HLA-E specifically may be needed to avoid an immune response from NK cells. Furthermore, immune checkpoint PD-L1 has been found to also suppress an immune response. Thus, maintaining or enhancing expression of HLA-E and PD-L1, in addition to inhibiting expression of HLA-ABC may provide a multi-tiered approach to avoiding the immune response for any allogenic CAR molecule.

Thus, described herein are novel RNAi DNA oligonucleotides for suppression of HLA-ABC, a method of making an allogenic chimeric antigen receptor (CAR) immune cell resistant to host cell rejection, and a method for increasing cytotoxicity of a chimeric antigen receptor (CAR) cell.

One embodiment described herein is an RNA interference (RNAi) oligonucleotide for inhibiting expression of HLA A, B and C (HLA-ABC). In one aspect of the embodiment, the RNAi DNA oligonucleotide comprises an siRNA or an shRNA. In another aspect, the RNAi DNA oligonucleotide comprises an short hairpin RNA (shRNA) that is conserved in common HLA-ABC alleles but has two nucleotide mismatches in the corresponding region in major HLA-E alleles. The shRNA is designed to specifically inhibit gene expression of HLA-ABC on immune cells, and thus reduce or eliminate surface HLA-ABC expression. The shRNA does not inhibit expression of HLA-E expression. Thus the, HLA-ABC reduced immune cells may escape allogeneic CD8+ T cell killing, HLA-ABC-specifically reduced, along with PD-L1 or HLA-E overexpressed immune cells may survive and escape allogeneic responses and controlled tumor progression in vivo. In another aspect the shRNA can also be applied to specifically knock down HLA-ABC in T cells, NK cells, induced pluripotent stem cells, or macrophages, or other cell types.